Continuous flow, preparative thin-layer chromatograph

ABSTRACT

An apparatus for thin-layer chromatography which comprises a support-coated glass plate, cover plates which are same in width as said glass plate and shorter in length than said glass plate and have spacers with same length on their both sides, clips for fitting said glass plate with said cover plate so that the latter plate can be moved up and down, and a solvent container with a lid into which the lower ends of both said glass and cover plates can be immersed.

United States Patent [56] References Cited UNITED STATES PATENTS 6/1965Brenner et al.

[72] Inventor Shoji llara Saitama, Japan 856,777

[2 1 1 Appl. No.

[22] Filed Sept. 10, 1969 [45] Patented Dec. 21, 1971 [7 3] AssigneeToyo Roshi Company, Limited Tokyo, Japan [32] Priority Sept. 27, 1968 lJapan ABSTRACT: An apparatus for thin-layer chromatography whichcomprises a support-coated glass plate, cover plates [54] CONTINUOUSFLOW, PREPARATIVE THIN- LAYlER CHROMATOGRAPH which are same in width assaid glass plate and shorter in 8 Claims, 10 Drawing Figs. length thansaid glass plate and have spacers with same length 52 their Sidesfitting Said glass with said 210,31 cover plate so that the latter platecan be moved up and down, 2105 198 and a solvent container with a lidinto which the lower ends of both said glass and cover plates can beimmersed.

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ATTORNEY I BRIEF SUMMARY OF THE INVENTION The present invention relatesto an improved sandwich-type apparatus for thin-layer chromatographywhich can separate and condense sample components in lines by thecontinuous flow evaporation and, more particularly, to an apparatus forthin-layer chromatography which comprises a support-coated glass plate,cover plates which are the same in width as said glass plate and shorterin length than said glass plate and have spacers with the same length onboth sides, clips for fitting said glass plate with said cover plate sothat the latter plate can be moved up and downduring chromatography, anda solvent container with a lid into which the lower ends of both saidglass and cover plates can be immersed.

An object of the invention is to provide an apparatus for thin-layerchromatography which is effected by a much shorter presaturation of thechromatographic chamber with solvent vapor and separates and condensessample components continuously at appropriate areas of thechromatographic plate without influencing the vapor saturation in thedeveloping chamber.

A further object of the invention is the provision of an apparatus forthin-layer chromatography which gives reproducible chromatograms and agood separation of the samples.

Another object of the invention is the provision of an apparatus forthin-layer chromatography which enables easy recovery of samplecomponents from a thin-layer plate by the elution with solvents.

A still further object of the invention is the provision of a thin-layerchromatographic apparatus of such nature that it prevents the solventdemixing which occurs in a thin-layer during the development when asolvent mixture containing polar solvents is used as a developingsystem.

A still further object of the invention is the provision of an apparatusfor thin-layer chromatography which is capable of rapid chromatographicdevelopment and minimizes the diffusion of sample spots separated duringthe development.

Other objects of the invention will be obvious from the contents of thespecification hereinafter disclosed.

CROSS-REFERENCES TO RELATED APPLICATION Various methods have beenproposed for preparative thinlayer chromatography of a sample mixtureapplied in line. However, they are not favorable in view of givingdiffused and distorted chromatographic bands of the sample components.For removal of these defects, Mulder and Veenstra have provided a deviceinvolving the evaporation of developing solvents from the space at thetop of the horizontal developing chamber, by which bands of the samplecomponents are resolved and narrowed. And then, they developed thechromatogram obtained thereby in the second-dimensional direction inorder to condense the bands as spots (J. Chromatogn, 24, 250 (1966) J.However, their method is not only so complicatedthat it could not bepractical, but itis difficult to condense the sample componentscontinuously. The continuous flow-developing method would be applicableto the box-type developing chamber proposed by Truter (J. Chromatogn,14, 57 (1964) as another device for example, However, it could not beutilized for the purpose of the stepwise condensation of the samplecomponents without placing a mount in the solvent container to raisecoated plate. Consequently, that may influence the magnitude ofsaturation of solvent vapor and result in worse resolution of thechromatographic bands. Change in the solvent vapor saturation during thedevelopment, in general, remarkable influences chromatographicmobilities of samples and poor resolution of mixture follows andfrequently inversion of their Rf values.

The sandwich-type developing chamber available on the market consists ofa cover plate with a spacer on its edge except the lower side, a glassplate with the same size as that of the cover plate and coated with anadsorbent layer except the area where the spacer contacts, and a solventcontainer. When it is used, the glass plate is fitted with the coverplate, and then the lower ends of both glass and cover plates areimmersed in a developing solvent places in the solvent container.However, slow soaking-up of the developing solvents and lowseparatobility of the sample mixture have given the apparatus muchdisadvantage.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS FIG. 1, FIG. 2and FIG. 8 show oblique views of another type in this invention and anenlarged sectional view at the line IVIv in FIG. 1, respectively.

FIG. 3 shows a cover plate on which a porous medium is attached andpressed down from both sides of the cover plate by slender springs.

FIG. 4 shows an oblique view of another embodiment of this invention andFIG. 5 and FIG. 8 show enlarged sectional views at the line VII-VII andVIII-VIII in FIG. 4, respectively.

FIG. 7 shows an electric heating means which is fixed on the glass plateso that the means can be moved up and down.

DETAILED DESCRIPTION OF THE INVENTION This invention overcomes theabove-mentioned defects, through an apparatus for thin-layerchromatography which includes a support-coated glass plate, cover plateswhich are same in width as the glass plate and shorter in length thanthe glass plate and have spacers with the same length as the cover plateon both sides and are used for covering the glass plate, clips forfitting the glass plate with the cove plate so that the latter plate canbe moved up and down, and a solvent container with a lid into which thelower ends of both said glass and cover plates can be immerses.

However, the chromatographic apparatuses have been found to causesolvent demixing in the adsorbent layer when solvent mixtures containingpolar solvents are used as developing systems. The above is based on thedifferent adsorptivities of the solvents used and leads to decrease inreproducibility of the chromatograms, giving different results fromthose obtained with the usual box-type chromatographic apparatuses.

The inventor has made further investigations to eliminate the solventdemixing and reached a conclusion that the defects mentioned above areovercome by attaching filter paper, glass fiber sheet, or any otherporous material, incapable of absorbing polar solvents, inside the coverplate not as to come into contact with the adsorbent layer and thenimpregnating it, prior to the development, with a solvent mixture to beused. Treatment of the cover plate as to give a porous inside is also aseffective as the above device.

The invention will be readily understood from the following detaileddescription.

Referring now to FIG. I and FIG. 2, the glass plate I is coated with anadsorbent layer on the inside. The cover plate 3, made of glass,plastics of metals whose width is same as that of the glass plate I andlength is shorter, has glass, plastic or metallic spacers 4'on bothsides which are thicker than the gel layer, viz 1-5 mm., preferably 2-3mm. The spacers 4 have even faces to prevent solvent vapor from leaking.By clips 5 the cover plate 3 is fitted on the gel-coated glass plate I,and their lower ends are immersed in a developing solvent places in acontainer 6. Loosening the clips 5, the cover plate 3 can be worked upand down at any appropriate position on the glass plate I. 7 is a lidfor the solvent container 6.

FIGS. 3, 4 and 5 also show an apparatus equipped with a porous medium 8attached between the spacers 4 shown in FIG. 1, which consists of filterpaper, glass fiber sheet, or any other porous medium 8 incapable ofadsorbing polar solvents. As shown in FIG. 4, for attaching the porousmedium 8 inside the cover plate 3, the use of spacers 4 with grooves intheir insides and pressing down the porous medium 8 by slender springs 9is recommended because they can prevent the porous medium 8 swollen withsolvents from slackening and coming into contact with the adsorbentlayer 2. Instead of the use of the cover plate 3 attached with theporous medium 8, a cover plate with a porous surface structure on itsinside face may be useful. In this case, there is no need to use thegrooved spacers. Thickness of the spacer should be 1-5 mm. morefavorably 2 to 3 mm.; thickness less than 1 mm. and more than 5 mm. isunfavorable because in the former, the developing solvent comes upthrough capillary action and in the latter, saturation of the adsorbentlayer with solvent vapor needs longer time.

FIGS. 6, 7 and 8 represent a cover plate 3 with spacers 4 having thesame thickness as those shown in FIG. 1, on both sides, and the numeralscorresponds to those in FIG. 1.

In FIGS. 1, and 6, the cover plate is equipped to cover the slant glassplate I faced upward, for convenience of explanation. The cover platemay also be used to cover the glass plate faced downward. In the lattercase, the porous medium attached inside the cover plate is preventedmore effectively from coming into contact with the adsorbent layer. Andalso the plates may be used vertically.

Furthermore, FIG. 7 shows an electric heating means 10 equipped on theupper side of the glass plate 1 of the opposite side of the cover plate3 as shown in aforesaid figures, so that the heating means 10 may beworked up and down at any appropriate position where sample componentsare condensed. The heating means 10 comprises a glass tube 11, anelectric heating wire 12 made of a nickel-chrome alloy inclosed in theglass tube 11, and the wire is connected to an electric source. Number13 shows adhesive tape retaining heating unit 10 on the glass plate I.

The operation, uses and advantages of the present device are as follows:

As an example of the operation and use of the present apparatus, thedevelopment of a mixture of three dyes is described (butter-yellow,Sudan-red G, and indophenol A solution of 2 mg. each of the three dyesin 0.1 ml. of benzene was applied in a line to the lower end ofa silicalgelcoated glass plate 1 (x20 cm. in size), and as illustrated in FIG.10a) the lower end of the plate 1 was then immersed in benzene placed inthe solvent container 6 after being covered with the cover plate 3(20x24 cm. in size) using clips 5. The developing solvent, benzene,migrated the dyes to give horizontally linear bands with differentmobilities during soaking up in the gel layer and was evaporated when itreached the position corresponding to the upper end of the cover plate3. Complete resolution of the dyes was achieved in 20 min. after thedevelopment, and the band of butter-yellow with the largestchromatographic mobility reached the top of the cover plate 3 and,according to the evaporation of the developing solvent, turned into avery narrow and condensed band about 40 min. later. After that the coverplate 3 was moved down to the position of 5 mm. underneath the condensedband of butter-yellow by taking off and refitting the clips 5, and thenthe development was continued as illustrated in FIG. 8). During theprocedure to move down the cover plate 3, little possibility of changein the magnitude of the vapor saturation was there, and consequently, nodisturbance was caused in chromatographic resolution of the dye bands.The band of Sudanred G was also developed to the top of the cover platein 4 hrs. and condensed as a very narrow band. Then the cover plate 3was moved down to the position ofS mm. underneath the condensed band ofSudan-red G, and the development was continued about 1 hr. more asillustrated in FIG. 8) until the band of lndophenol reached the top ofthe cover plate and was condensed.

While only one embodiment of the device has been described in detail, itis particularly understood that the device is not limited thereto andthereby, but can be applied to the resolution of mixtures consisting ofmore than three components. In this case, when the lower end of theglass plate I can not be immersed in the solvent by moving the coverplate 3, the latter plate is replacedwith a shorter one for furtherchromatographic procedures.

As a summary; (1) The recovery of each component of a mixture is veryreadily established by making use of the present apparatus. (2) Samplescan be chromatographed without presaturation of the thin-layer platewith solvent vapor, and which does not cause any disturbance of theirchromatographic mobilities, permitting fairly good reproducibility ofthe results and easy operation. (3) It is easy to alter the area forcondensing the sample band without disturbing the saturation of solventvapor. (4) The apparatus can be utilized not only when any type ofchromatographic supports coated in various thickness are used, but alsorather efficiently for the usual purpose of thin-layer chromatographybecause of its advantage that the presaturation with solvent vapor isunnecessary.

Referring again to FIG. 1 for further explanation about the practicaluse of the present apparatus, after application of a solution of samplemixture to the lower side of the thin-layer 2 coated on the glass plate1, the cover plate 3 fitted with a porous medium 8 is equipped usingclips 5 to cover the adsorbent layer, and prior to the development, onlythe lower end of the porous medium 8 is immersed in a developing solventmixture places in the solvent container 6 to saturate the thinlayer 2with solvent vapor. Though the time needed for the saturation differsaccording to nature of the solvent mixture used, it is usually only afew minutes. After that, the cover plate 3 is moved up a little byloosening the clips 5 and then tightening them, the both ends of theglass plate 1 and the cover plate 3 are immersed in the solvent mixture.The solvent mixture migrates the components of sample during soaking upin the thin layer and is evaporated after reaching the top of the coverplate 3. After an appropriate interval, the band of the component withthe largest mobility reaches the top of the cover plate 3 and condensesthere in a line.

After the complete condensation of the first developed band, the clips 5are loosened and the cover plate 3 is moved down at a small distancefrom the line of the first component. Tightening the clips 5, thedevelopment is continued until the second component is condensed at thetop of the cover plate 3. Repetition of the above procedure leads allcomponents of the mixture to the complete separation and condensation inlines at appropriate positions.

The apparatus illustrated in FIG. 4 used in the same way as describedabove for the one in FIG. 1. Prior to the development, the cover plate 3is fitted on the glass plate 1 with the clips 5 so that only the lowerend of the porous medium attached inside the cover plate can be immersedin the developing solvent mixtures. The time needed for the saturationwith solvent vapor is almost same as in the former apparatus. Afterimmersion of both ends of the cover plate 3 and the glass plate 1 in thesolvent mixture, the development is carried out. According to soaking upof the solvent in the thin layer, the sample applied to the lower sideof the layer is chromatographed.

The use of the present apparatus as shown in FIG. I and 4 gives moreadvantages in comparison to the ones available on the market. From theviewpoint of accelerating the evaporation of less volatile solvents, afurther advantage can be obtained by the use of the heating device shownin FIG. 7.

a. They establish rapid development, prevent diffusion of spots or bandsof the sample components, and give wellresolved chromatograms with highreproducibility.

The above will be demonstrated by the following examples which wereobtained with a silica gel thin-layer plate with a thickness of250uprepared in the usual manner. The development was carried out 10 cm.from the lower end of the plate.

Time for 10 cm. development (min.) Solvent Solvent C H.Me,CO(4:l) CH.-MeOH(9:l) The present apparatus 18:2 19:2 in FIG. I The presentapparatus 21:2 2212 A usual sandwich type 29:2 29:2 apparatusChromatography of a mixture of Nehers dyes in a solvent mixtureC,,l-l,,-MeOH(9:l) is shown below. ln this experiment, the same lot ofsilica gel (Wakogel B) was used for preparation of the thin-layer with athickness of 250 4., and the development was carried out 10 cm. from thelower end of the plate.

Making use of the present apparatus, samples applied are chromatographedto give smaller and less diffused spots compared with the use of usualsandwich type apparatus.

Chromatographic mobilities (RfXlOO) and their standard deviation(SDXlOO) of the dyes, obtained with 10 experiments, are shown in thefollowing table. The data listed indicate that the present invention issuperior in the reproducibility of the results.

The apparatus in Fig. 1

Rf 100 99, 79, 68, 53, 45, 37, 41, 34, 28 SD 100 1, 1,1,1,1,1,1,1,1

The apparatus in Fig. 4

RfX100 99, 80, 69, 53, 40, 36, 40, 33, 29 SD 100 1, 2, 1, 1, 1, 1, 1, 1,1 The usual sandwich type apparatus RfX 100 99, 60, 55, 46, 38, 36, 40,37, 36 SD 100 1, 3, 2, 3, 4, 2, 1, 2, 1

b. Solvent mixtures containing polar solvents cause littleC,H.Me,CO(4:l) 0.70-0.83 C,H MeOH (9:l) 0.20-0.33 CHCl,-Me,CO(3:l)0.86-0.92 Hexane-EtOAc (2:8) 0.96-0.98 CHCl ,Me0H (97:3) 0.31 CHCI -HOAc(3:1) 0.75

Not only is the B-front not observed with the present apparatus whichhave a porous medium inside the cover plate, but also they give muchbetter separability of the sample components. This is based on anadsorption effect of the vapors of polar solvents which comes from theporous medium attached inside the cover plate.

What is claimed is: 1. An apparatus for effecting thin-layerchromatography which comprises:

a. a coated support plate, wherein said coating comprises a thin-layerof adsorbent material; I

b. a cover plate for said support plate having a comparable width,wherein said cover plate has a porous medium disposed on the surfaceadjacent to the thin-layer of the coated support plate;

c. a means for maintaining said cover plate parallel to and apart fromsaid coated support plate and d. a solvent container in which the bottomedge of said support plate and said cover plate is emerscd and exposedto solvent.

2. The apparatus of claim 1, wherein the means for maintaining saidcover plate parallel to and apart from said coated support platecomprises spacers placed between and extending the length of the edgesof said plates so as to minimize solvent vapor loss.

3. The apparatus of claim 2, wherein said means comprise spacers havinga thickness of from about 1 to about 5 millimeters.

4. The apparatus of claim 2, wherein said means comprise spacers havinga thickness of from about 2 to about 3 millimeters.

5. The apparatus of claim 1, wherein the porous medium disposed on thesurface of said cover plate is selected from the group consisting offilter paper, glass fiber and fiber sheet.

6. The apparatus of claim 5, wherein said porous medium is im re natedwith solvent prior to use, i

. e apparatus of claim 5, wherein said porous medium is retainedadjacent to said cover plate by means of slender springs.

8. The apparatus of claim 1, wherein a movable heating means is disposedon the remote surface of said cover plate.

2. The apparatus of claim 1, wherein the means for maintaining saidcover plate parallel to and apart from said coated support platecomprises spacers placed between and extending the length of the edgesof said plates so as to minimize solvent vapor loss.
 3. The apparatus ofclaim 2, wherein said means comprise spacers having a thickness of fromabout 1 to about 5 millimeters.
 4. The apparatus of claim 2, whereinsaid means comprise spacers having a thickness of from about 2 to about3 millimeters.
 5. The apparatus of claim 1, wherein the porous mediumdisposed on the surface of said cover plate is selected from the groupconsisting of filter paper, glass fiber and fiber sheet.
 6. Theapparatus of claim 5, wherein said porous medium is impregnated withsolvent prior to use.
 7. The apparatus of claim 5, wherein said porousmedium is retained adjacent to said cover plate by means of slendersprings.
 8. The apparatus of claim 1, wherein a movable heating means isdisposed on the remote surface of said cover plate.